Dual targeting of a virus movement protein to ER and plasma membrane subdomains is essential for plasmodesmata localization

<div><p>Plant virus movement proteins (MPs) localize to plasmodesmata (PD) to facilitate virus cell-to-cell movement. Numerous studies have suggested that MPs use a pathway either through the ER or through the plasma membrane (PM). Furthermore, recent studies reported that ER-PM contact sites and PM microdomains, which are subdomains found in the ER and PM, are involved in virus cell-to-cell movement. However, functional relationship of these subdomains in MP traffic to PD has not been described previously. We demonstrate here the intracellular trafficking of fig mosaic virus MP (MP_FMV) using live cell imaging, focusing on its ER-directing signal peptide (SP_FMV). Transiently expressed MP_FMV was distributed predominantly in PD and patchy microdomains of the PM. Investigation of ER translocation efficiency revealed that SP_FMV has quite low efficiency compared with SPs of well-characterized plant proteins, calreticulin and CLAVATA3. An MP_FMV mutant lacking SP_FMV localized exclusively to the PM microdomains, whereas SP chimeras, in which the SP of MP_FMV was replaced by an SP of calreticulin or CLAVATA3, localized exclusively to the nodes of the ER, which was labeled with <i>Arabidopsis</i> synaptotagmin 1, a major component of ER-PM contact sites. From these results, we speculated that the low translocation efficiency of SP_FMV contributes to the generation of ER-translocated and the microdomain-localized populations, both of which are necessary for PD localization. Consistent with this hypothesis, SP-deficient MP_FMV became localized to PD when co-expressed with an SP chimera. Here we propose a new model for the intracellular trafficking of a viral MP. A substantial portion of MP_FMV that fails to be translocated is transferred to the microdomains, whereas the remainder of MP_FMV that is successfully translocated into the ER subsequently localizes to ER-PM contact sites and plays an important role in the entry of the microdomain-localized MP_FMV into PD.</p></div

PD localization of MP_FMV is necessary for their cell-to-cell movement.

<p>Investigation of the ability of MP_FMV and its mutants (MP_FMV:YFP, Trun:YFP, SP_spoTrun:YFP, SP_calTrun:YFP and SP_clvTrun:YFP) to move to adjacent cells. (A) Typical images were captured at 3 dpi. Bars = 50 μm. (B) Quantitative analysis. The bars show means + SD. n indicates the total number of measurements in two independent experiments. Different letters on error bars indicate statistical differences at the 1% level of significance (Tukey-Kramer test).</p

MP_FMV has an N-terminal signal peptide.

<p>(A) SignalP ver 4.1 predicted an N-terminal signal peptide in the MP_FMV sequence. C-score is the predicted cleavage site value, S-score is the predicted signal peptide value and Y-score is the cleavage site value calculated by combining C- and S-scores. (B) Immunoprecipitation of MP_FMV:FLAG. MP_FMV:FLAG was immunoprecipitated with anti-FLAG antibody from cell lysates of healthy control leaves (lane 1) or of MP_FMV:FLAG expressing leaves at 48 hpi (lane 2). The immunoprecipitated samples were checked by immunoblot analysis using anti-FLAG antibody (left panel) and Coomassie Brilliant Blue (CBB) staining (right panel). The lane between lane 1 and 2 is blank in CBB staining. (C) Hydrophobicity of the predicted SP sequence. Amino acid residues are color-coded according to their hydrophobicity. The dotted line with scissors indicates the putative cleavage site.</p

A model for the intracellular trafficking of MP_FMV.

<p>A model for the intracellular trafficking of MP_FMV.</p

ER translocation efficiency of SP_FMV differs from those of plant protein SPs.

<p>(A) Schematic representation of fusion proteins for investigating ER translocation efficiency. (B) Cells expressing free GFP, GFP:HDEL, SP_FMVGFP:HDEL, SP_spoGFP:HDEL, SP_calGFP:HDEL and SP_clvGFP:HDEL were observed at 36 hpi. Bars = 10 μm. (C) Schematic representation of the experimental system and fusion proteins with a 3×N-glycosylation sequon. Dots indicate asparagine residues expected to be glycosylated. SPs of MP_FMV, sporamin A, calreticulin or CLAVATA3 were fused to the N terminus of GFP:glc, GFP carrying a 3×N-glycosylation sequon in its C terminus (SP_FMVGFPglc, SP_spoGFPglc, SP_calGFPglc and SP_clvGFPglc, respectively). (D) Immunoblot analysis of proteins extracted from cells expressing GFPglc, SP_FMVGFPglc, SP_spoGFPglc, SP_calGFPglc or SP_clvGFPglc using anti-GFP antibody. Samples were collected at 30 hpi. Glycosylation of each sample was confirmed by deglycosylation with Endo H. (E) Quantitation of translocation efficiencies. The bars show means + SD of three independent experiments. Different letters on the error bars indicate statistical differences at the 1% level of significance (Tukey test).</p

Properties of MP_FMV and its mutants.

<p>Properties of MP_FMV and its mutants.</p

Investigation of the subcellular distribution of MP_FMV:YFP.

<p>(A–C) Confocal imaging of MP_FMV:YFP-expressing epidermal cells at 36 hours post-infiltration (hpi). YFP fluorescence was pseudocolored by magenta. (A) (i) Cells expressing MP_FMV:YFP were treated with aniline blue. Arrowheads indicate plasmodesma. The mean ± SD of Pearson correlation coefficient (PCC [–1:1]) is given in the image. Bars = 5 μm. (ii) Fluorescence intensity along the arrow across plasmodesma. The dotted line in the confocal image indicates the cell wall. Bar = 1 μm. (B) Plasmolyzed cells expressing (i) MP_FMV:YFP or (ii) 30K:GFP. Arrows indicate Hechtian strands extended from the PM. Arrowheads indicate plasmodesmata. The dotted line indicates the cell wall. cw, cell wall; cyt, cytoplasm. Bars = 10 μm. (C) Surface views of cells (i) expressing MP_FMV:YFP or (ii) treated with FM4-64. (iii) Co-expression of MP_FMV:CFP and YFP:REM1.3. To obtain higher resolution images, images were processed by a deconvolution algorithm. Bars = 5 μm. (D) 1% TritonX-100 treatment of membranes. Membrane-enriched fractions prepared from FMV-infected fig leaves and <i>N</i>. <i>benthamiana</i> leaves expressing MP_FMV:FLAG or YFP:REM1.3 at 36 hpi were treated with 1% TritonX-100. Anti-FLAG, anti-MP_FMV, anti-GFP, anti-H⁺ATPase and anti-BIP antibodies were used for the detection of MP_FMV:FLAG, MP_FMV, YFP:REM1.3, a PM marker H⁺ATPase and an ER marker BIP. S; soluble fraction. P; insoluble fraction.</p

ER-translocated MP_FMV specifically localizes to ER-PM contact sites.

<p>(A and B) ER-translocated MP_FMV co-localized with SYTA, a protein localized to ER-PM contact sites. 3D-projection images of cells expressing SYTA:CFP and (A) SP_clvTrun:YFP or (B) MP_FMV:YFP. Z-section images of 10 slices at 1.0 μm intervals were processed. Arrowheads indicate co-localization of SYTA:CFP and MP_FMV:YFP. (C and D) Expression of SYTA^ΔC2B, a dominant-negative form of SYTA, affected MP_FMV localization. (C) SYTA^ΔC2B:myc was co-expressed with MP_FMV:YFP. Plasmodesmata were visualized by aniline blue treatment. (D) SYTA^ΔC2B:myc was co-expressed with MP_FMV:YFP and SYTA:CFP. A cortical region was visualized. (A–D) Cells were observed at 36 hpi. YFP fluorescence was pseudocolored with magenta. Bars: (A–D), 5 μm; (C) inset 2.5 μm. (E) SYTA^ΔC2B:myc showed an inhibitory effect on MP_FMV movement to adjacent cells. MP_FMV:YFP was co-expressed with GUS or SYTA^ΔC2B:myc. (i) Typical images were captured at 3 dpi. Bars = 50 μm. (ii) Quantitative analysis. The bars show means + SD. n indicates the total number of total measurements in two independent experiments. The asterisk above an error bar indicates a statistical difference at the 1% level of significance (Student's t-test). (iii) Immunoblot analysis using anti-myc antibody (top panel). CBB staining is shown as a loading control (bottom panel). Samples were collected at 36 hpi.</p

SP-deficient MP_FMV localizes to PD when co-expressed with MP_FMV or SP chimeras.

<p>Trun:CFP was co-expressed with (A) MP_FMV:YFP, (B) Trun:YFP, (C) SP_spoTrun:YFP, (D) SP_calTrun:YFP or (E) SP_clvTrun:YFP. Cells were observed at 36 hpi. YFP fluorescence was pseudocolored with magenta. Arrowheads indicate localization to PD. Bars in images of detailed view are 2.5 μm. The others are 10 μm.</p